Recent evidence suggests that alterations in beta-cell gene expression may lead to the beta-cell defects seen in non-insulin dependent diabetes mellitus (NIDDM), the most common form of diabetes. The experiments proposed here will directly test this hypothesis. The information and reagents provided by these investigations will contribute ultimately to the understanding, treatment and cur of diabetes. Our long term objective is to determine how glucose and other nutrients regulate gene expression in the beta-cells of the pancreatic islets of langerhans and to apply this knowledge to understanding the pathophysiology of NIDDM. Our general strategy is to focus on the insulin promoter and use it as a probe to dissect the transcriptional machinery in normal beta-cells. An important aspect of this work is the use of normal beta-cells in primary islet cultures as opposed to aberrantly regulated tumor cell lines. Our ability to transfect cultured islets allows us to study gene expression in both normal beta-cells and diabetogenic beta-cells from ZDF rats. Our specific aims are directed at dissecting the mechanisms by which glucose regulates the insulin promoter. These experiments will lead to the identification and characterization of the transcription factors that regulate the promoter in response to metabolic signals. We will focus on the transcription factors because we believe that the transcription factors and their proximal regulators are the most likely targets for defects in NIDDM. The first aim of this proposal is to carefully delineate what sequences within the insulin promoter code for the response to glucose, and how these sequence elements interact. In the second aim, the proteins that bind these sites will be identified and characterized; and in the third aim, we will investigate how glucose regulates the previously identified Pan transcription factors. The signaling pathways by which nutrient molecules regulate these factors will be tested in the fourth aim. We will focus on the calcium signaling pathway because of preliminary evidence supporting a role for this pathway in glucose regulation of insulin gene transcription. The knowledge of gene expression in normal beta cells developed in the first four specific aims will provide an unique opportunity to achieve our final aim, characterization of beta-cell gene expression in the ZDF rat model of NIDDM. Our preliminary data suggests that these animals have a defect in beta-cell gene expression, and our experiments are designed to localize this defect.